A major objective of The Section on Molecular Neuroscience is to discover the components of non-canonical cAMP signaling initiated by PACAP that mediate stress-induced gene transcription at central and peripheral synapses. We have employed cDNA microarray to identify groups of gene transcripts induced by PACAP in cell culture models in vitro, and induced in wild-type but not PACAP-deficient mice, in stress paradigms in vivo. Treatment with PACAP of cultured chromaffin cells of the adrenal medulla itself, or PC12 cells, a chromaffin-like continuous cell line, models the post-synaptic effects of PACAP upon release from neurons in vivo. We have demonstrated with microarray analysis (Eiden et al., 2008) that PACAP induces neuroendocrine-specific genes in PC12 cells in early (1-3 hours), intermediate (3-6 hours) and late (6-48 hours) phases. Early regulated genes include transcription factors such as Nur77, Nurr1, Egr1, and Fosl1. Intermediate genes include several that regulate calcium homeostasis. Late regulated genes include those encoding neuroendocrine secretory proteins, e.g. chromogranin B. However, neuropeptides such as galanin, VIP and substance P, induced in chromaffin cells by splanchnic nerve stimulation in vivo or by PACAP treatment of bovine chromaffin cells in vitro, were not induced by PACAP in PC12 cells. PC12 cells are a limited model for chromaffin cell regulation by PACAP, because the PAC1 receptor is present at only low levels in PC12 cells, and responds to PACAP with a large increase in intracellular cAMP, but only a weak calcium influx response compared to chromaffin cells. A PC12 cell line was created that stably expressed levels of the PAC1hop isoform of the receptor comparable to those present in chromaffin cells. In the PC12-bPAC1hop cell line, PACAP treatment causes a prolonged elevation of both cAMP and calcium influx, and concommitant up-regulation of genes encoding neuropeptides such as substance P. A full microarray study of gene induction in PC12 compared to PC12-bPAC1hop cells revealed additional genes, besides those encoding neuropeptides, that were uniquely induced in the latter cell line, and have tentatively been assigned roles in modulation of intracellular calcium under conditions of metabolic stress. [unreadable] We next carried out studies of gene transcription in the adrenal gland after splanchnic nerve stimulation following insulin injection (insulin shock metabolic stress model). As in PC12 cells treated with PACAP, immediate early genes including Egr1, Nur77 and Nurr1 were highly induced in the adrenal gland within 1-2 hours of hypoglycemic shock. Neuropeptide genes were induced somewhat later. Shock-induced gene expression in mice deficient in PACAP resulted in a significant attenuation of neuropeptide (galanin, VIP and substance P, or Tac1) gene induction, but, surprisingly, no alteration in induction of the immediate-early genes, although these were PACAP-responsive in vitro (Stroth, Hamelink and Eiden, in preparation). PACAP and acetylcholine, the slow and fast transmitters released at the adrenomedullary synapse in vivo, therefohave redundant functions to mediate immediate-early gene induction, while PACAPs unique role is to mediate the excitation-dependent cellular plasticity (long-lasting up-regulation of neuropeptide expression) which is a hallmark of the adrenal response to prolonged stress. We had previously obtained evidence that induction by PACAP of one of these genes, VIP, relies upon the non-canonical cAMP pathway (Hamelink et al., J. Neurosci. 22:5310, 2002). Thus, the non-canonical pathway appears to have physiological relevance at the adrenomedullary synapse, and the bovine chromaffin cell will provide an accessible primary neuroendocrine cell model in which to explore this novel signaling pathway mechanistically.[unreadable] PACAP can activate the non-canonical signaling pathway in PC12 cells, which also contain a well-characterized cAMP-PKA signaling pathway. PC12 cells had been used by other laboratories to make the important observation that cAMP signaling to activate the MAP kinase ERK1/2 required PKA-dependent activation of B-Raf through the small GTPase Rap1. This pathway is critical for induction of such neuron-specific genes as transin, and the protein comprising the sodium channel. We used our extensive collection of PACAP-responsive genes derived from microarray analysis to screen target genes sensitive to inhibition of ERK, and those sensitive to inhibition of PKA. Activation of neurite formation, induction of Egr1, and induction of the stress response protein gene Ier3, were ERK-dependent and PKA-independent, while induction of ornithine decarboxylase was ERK-independent, and PKA-dependent. Use of siRNA for these gene targets, and a dominant negative expression vector for Rap1, allowed us to make the important observation that PACAP initiates a cAMP-dependent, PKA-independent signaling pathway through Rap1 and ERK that activates Egr1 leading to a critical neuronal function, neurite extension (Ravni et al., 2008). Thus, two cAMP pathways activating ERK, one requiring PKA and one not, exist side-by-side in PC12 cells, mediating complementary roles which are both necessary for neuronal function. We have further observed that activation of Ier3 gene expression by PACAP can be mimicked by forskolin, in cells which are PKA-clamped by treatment with the PKA inhibitor H89, only upon co-treatment with KCl, which causes cell depolarization and increased calcium influx. These data (Eiden, Holighaus and Gerdin, in preparation) strongly suggest that the cAMP sensor activated for PACAP signaling to ERK requires co-stimulation by calcium. PACAP-dependent gene transcription also proceeds through activation of the ERK pathway in rodent cortical neurons and in neuroblastoma-glioma hybrid (NG108-15) cells expressing the hop variant of the PAC1 receptor (Holighaus, Gerdin, Liu and Eiden, unpublished observations), indicating that this neuropeptide-dependent signaling pathway occurs throughout the nervous system.[unreadable] Activation of calcium signaling by PACAP highlights a second unique aspect of PACAP neuroendocrine gene regulation. We and our colleagues at the University of Rouen have observed that bovine chromaffin cells express the type 2 TNF receptor, shown by others to be linked to neuroprotective effects of TNF in the CNS. In chromaffin cells, TNFR2 mediates persistent NF-kB signaling (Ait-Ali et al., 2008) leading to neuropeptide gene induction (e.g. VIP and galanin) which is potentiated by PACAP. Adrenal VIP is induced in wild-type but not PACAP-deficient mice in the LPS sepsis model (Ait-Ali et al., in preparation). Activation of NF-kB, leading to potentiation of TNF signaling, could play an important role in PACAP anti-inflammatory effects in the CNS. NF-kB activation may also be involved in PACAP induction of the novel anti-apoptotic protein serpinb1a, since the induction of the latter is blocked, along with PACAP neuroprotection of PC12 cells upon serum withdrawal, by inhibition of calcineurin. Calcium-dependent, i.e. BAPTA-AM-sensitive, effects of PACAP on gene transcription in PC12 cells are not blocked by inhibitors of phospholipase C or calcium channel blockers, suggesting that classical pathways to calcium-dependent gene activation are not used by PACAP in the context of gene regulation (Eiden,Holighaus and Gerdin, in preparation). Rather, a unique mode of regulating cytosolic calcium, occurring upon activation of the PAC1 receptor in neuroendocrine cells,drives gene regulation by this neuropeptide.[unreadable] In the coming year, the Section will continue investigation of PACAP target gene regulation in CNS neurons as well as chromaffin cells, and biochemical analysis of the unique PACAP-operated cAMP and calcium signaling pathways that control transcription in homeostatic neuroendocrine stress responses.